Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1936 Cytological studies of certain apple varieties and their seedlings, with special reference to their value as stocks Samuel Wheeler Edgecombe Iowa State College

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Recommended Citation Edgecombe, Samuel Wheeler, "Cytological studies of certain apple varieties and their seedlings, with special reference to their value as stocks " (1936). Retrospective Theses and Dissertations. 13506. https://lib.dr.iastate.edu/rtd/13506

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GYTOLOGIOAL STUDIES Of CERTAIN APPLE VAIilSTIES -m THEIH

SEIDLIEGS vJTH SPSOIAL BSfERSNOS TO THEIR YALT31 AS STOCKS

BY

Samuel Wheeler Bdgeeoabe

A Thesis Submitted to the Grafiuate faculty for the Degree

i30CTOR 0? HilLOSOPEY ^

Major Subject Horticulture

Approved:

Signature was redacted for privacy. In Giiarge of Major woric

Signature was redacted for privacy. Head of Major Departaent

Signature was redacted for privacy. Dean of Graduate College.

lovm 'Sta'te"Ooliege 1936 UMI Number: DP13193

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UMI Microform DP13193

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TABLE OF COKTELITS

Page

Introduction 4

Purpose of Investigation 19

Re'uiew of Literature SO Table 2. List of chrcMoscme numuers in horticultural epple varieties 27 Table S. List of cJiroiaosome numbers in apple species 35

Ixperiraental 38 i.uitorials 38 Methods 29 Cytological investigatious 41 •Whitney (3n) 41 Araes 550 (ga) 42 Virginia Crab (Srs,) 45 Hibernal {Zn) 44 MisDJTi {8n) 45 Starlcing (2n) 46 Delioious (2n) 46 King Davis (Sn) 4? Jonathan {Sn) 47 Stajr-nan (iJn) 47 Ames 541 (Sn) 48 (Jriines (£n) 4S Table 4. Cori^jarison of obserTecl nornial and "abnorrsal" first division, anaphcse figures in diploid and triploid var­ ieties 50 Table 5. Comparison of observed noruial and "abr).or:uc.l" ' sesoiid civlsion aiiaphi.;Ge figures in diploid and triploid var­ ieties 31 Table 6. Comparison of the nuraber and percentage of empty and "normal" or full polien f-rains 'felthin the anthers of .acies 550, Whitney, Hibernal and Virginia Crab 5S Fruit setting in reciproco.1 croEses involving the triploid varieties, Hibernal and Virginia Crab 53

rH84 TABLE-OF C0KT1-HT3(continued) Page

Table 7. ]?ruit setting in crosses between 'fthitney $ , and Aiaec 350 , Dxicliess

Msoussion and Oonclusions 59 O:iirojriosome constitution of cliploida 5S C.liro:rioso.rae constitution of triploids 59 Pollen studies of diploid and triploid vsrieties 60 Fruit setting in reciprocal crosses involving tb.G triploid varieties, Hibernal and Virginia Crab 62 General 6S

Summary 67

Explanation of plates 59

Literature Oitefi 79

Acknowle nt s 83 IKTfiODUCTIOH

Araerlcan nursei'jaiien heve depended largely upon the so- oalled ]?rencli Crab, either /oaerioan or French grovm, for tlieir seedling apple stocks although soiae Veraont and. Korthern grovai seedlings have been used. Apple stocks are nov^ bein^^ jiroduced in considerable quantities by Oregon and '!:^•asl:.lingtOE nursery­ men. Hone of these seedling stoclcs are entirely satisfactory in the uxn)er Mississippi Valley because they laclc sufficient hardiness to x^ithstancl the severe winter teeiperatures of thet region.

The PoHiOlogy Subsection, lov'/a State College, iliues, Iowa, is maMng efforts to find stocks which v;ill be luore satis­ factory than those now used in the upper Mississippi Valley,

The three lines of approach vMch are being follov/ed are:

(1) 1'he development of own-rooted, stoclcs, (S) Stocks grorm from open-pollinated seeds from selected pcirents and, (5)

Double Y^orked, stocks in which an intermediate stem .piece is insartecl between the original root stock and the top-wor3cec( variety. (Maney 25, S6, 87, 88, and 39),

Certain vigorous herdy varieties, such ss, Hibernal and

Virginia Crab, when used as seed parents in the Io\Ma studies, produced open-pollinated seedlings Vfhich were so lacking in vigor that they were vdthout value for stocks. (Haiiey 26 and 27). On t'ae other hand, vc-rieties suoh as, Whitney and Aaias

550^, tvhich are medium vigorous varieties, produced open- pollinatefi seedlings, \v'hich Vi-ere very vigorous and excellent for stock purposes.

After Mane;^' (27) observed the striidng "beha-viour of these four varieties, he grew open-pollinatee. seedlings of them for four successive seasons. The seed \'?as eollected froiii various locations, so th-ot each variety was likely to have been pollinated by a wide range of pollenlxers. Ho^vever, regardless of the year or place of collection Maney (28) found that the two varieties, HiDeraal and Yirginia Crab, produced oeedlings laolcing in vigor v.hile the other tvfo varieties, Whitney and Mes 550, produced seedlings which v/ere strong and vigorous, (iklso see figures 1-3).

Further, in his b?>.-eeding investigations, Lantz (20) of the same station, has had great difficulty in malcing crosses using Hibernal, (Unpublished data). When Hibernal was used as the pollen parent, the crosses y.ere complete failures. i'l'hen used as the pistillate parent an occasional fruit v/as obtained; but such fruits usually had very few seeds. These seeds germinated poorly and finally the fm resulting seed-

1 Araes 550 is a seedling produced by the Pomology Subsection, Iowa State College, i\jTies, lov/a, from the cross, Briar Sweet X. Mercer County Grab. Figure 1. Tlie apple seedlings in the row indicated by the arrow are character­ istic of one year old Virginia Grab seedlings. Note the poor stand and lack of vigor of this row of seedlings as compared v/ith the seedlings in the adjoining rows 2. T1i8 row indicated, by "fclie arrow of one year old Hibernal seedlings show the type of stand and vis^or which may be expected when Hibernal is used as a seed parent. Figxire 3. Comparison of five year old open-pollinated seedlings of Virginia Crab, Ames 560, and Stench. Crab. The Virginia Crab seedlings are in the rov/ indicated by the arrow, the seedlings in the row on the left are Ames 550, and those on the right are French Crab seedlings. Table 1. STJHMMiY OF iiiJTONOVKA AKE lilBERKAL PkOGSTIISS IN Tfffi SEISDETX SEOVJIKG mmBBU OF SEF.D3 PLmTEV, mjMBER j\ND FEl.OEKTAG-E OF aSI^DS GESiaNATIKG, KUMBSE OF SSEDLiSGS iJYIKG AT TIE EKD OF THE I'lEST TEAK'S GRO'JTH, AND THIS ATSRAGS II2IGHT OF THB SSSDLIHGS IN E/,.CH tROOEKY AT TEE iKD OF THE FIRST YlSiiR'S GRGYITH. :lJo. seedB: :Average ht. Parentage Gorniination results Breedin^r : : : of -pToa.BXiY N-uiaber Nusaber : Percentage • in inches

14210 •Axitonovlca X Delicious 375 S71 72.27 9.88 14246 t? X Delicious 87 64 73.56 7.38 It 1421£ x Blaok Oxford 238 200 84.03 9.17 14E08 f? X Bleclc Oxfoi-d 27 12 44.40 7.90 14211 f-r 3£ Ashton 200 111 55. 50 8.35 14S40 T? X Griraes 105 63 60 .00 7.08 t? X Jonathan 115 84 73.04 6,12 14S13 ?f 14215 X King David 163 15 S 87.36 8.3©

14S04 Hibernal X Ashton 29 13 44.83 5.58 14221 tt X Black Oxford 106 55 51.88 S.60 14218 r? X Delicious 75 30 41.10 4,00 142E3 It X King David 113 25 20.36 3.31 t» 14E2E 2<. Northern Spy £7 4 15.OO 4.00 14254 ft X Delicious 20 5 25.00 4.40 14318 ?f IX Jjelicious 5 1 20.00 5.00 14234 n X Jonathan S 2 66.67^ 0.00* 14522 tp X Delicious 57 14 57.85* 0.00-"^ 14237 tt x J onathan 11 0 0 . 00 0.00

'''The average height of theae seedlings was measux-ed at the end of the first year's growth. By that time all of the seedlings in these progenies were dead. These data are aocording to l^antz (30). - 10 -

I » t J iai>

Flgxire 4. Hibernal, x. Ashton. (5n x 2n). This cross­ bred apple seedling is eleven years from seed. Figure 5. Hibernal x Asliton. (3n z 2n?). This crossbred apple seedling is eleven years from seed. - 12 -

Figirre 6, Hibernal x King David. (3n z 2n). This crossbred apple seedling is tMrteen years frota seed. - 13 -

Figure 7, Hibernal z Black Oxford, (3n x 2n?), This crossbred apple seedling is thirteen years from seed. - 14 -

Figure 8. Hibernal x Jonathan. (3n x 2n), This crossbred apple seedling is eleven years from seed, Figure 9. Hibernal x Delicious, (3n x 2n). This crossbred apple seedling is ten years from seed. - 16 -

yigiore 10. HilDernal z Esopus. • (3n 2; 2n). This orossljred apple seedling is ten years from seed. - 17 - lings were alv;ays lacking in vigor throuf^hout their lives.

In fact, only rarely have tiiey reached fruitiag age or sise.

{See figs. 4-10 and TalDle I).

Other workers, BeauDSont (S) and the eAperiiaental farm at Lern-iosville, Quebec (S3) have reportea that Hibernal has produced seedlings of desirable vigor. This apparently con­ flicting evidence should not be talcen as such, because Gibb

(11, 12) aM Lyon (24) have shown that Vvhen the various

Russian apples vere introduced into Merica, several strains vjere nsoned. Hibernal. In view of the ciiaraetrically opposite results observed it is jiossible that the clones used by the different investigators are not the same.

Until 1930, the laultiple factor hypothesis y!8.a used to explain the lack or presence of desirable vigor in seedlings of apple varieties. At that time Crane and Lav-rence (4)

Yi'orking in close co-opei'ation vdth Darlington and Moffett (7) advanced another explanation for auch csvsos of extreme lacli of vigor as were observed by Maney (26) vdth the seedlings of

Hibernal and Virginia Crab, They hed observed a variety,

Braraely's Seedling, ^vhich behaved In much the same manner as

Hibernal and Tirginia Grab, in that it produced seedlings lacking i.n vigor. This variety v/as investigated cytological- ly by Dsrlington and Moffett (7) and found to be a triploid form ?dth 51 cliromoyoffies. Furthermore, IZ open-pollinated - 18 - seedlings of Brarnely's Seedling were e7caiained cytologioally.

All were aaeuploids with cliromosorae coimts ranging frora 36 for the lo'vest to 47 for the highest in the series. On the basis of these data they adiraiiced the theory that crosses betv/een triploid and diploid apple varieties yield only aneuploid seedlings, T.'hich are lacking in vigor d.ne to their aneuploid chroinosoMS constitution.

Crane and Larvrenoe (4) at the same time reported the results of nine crosses involTing diploid x diploid, diploid

X triploid and triploid x triploid apple varieties. Onl.y seedling vigor obser^^ations were made but the evidence tended to confirm Darlington and Moffett's theory. Also, results obteined by Dahl (6) v;ere in a^vreement with the theory. Up to the time of the initiation of this investigation this vms the situation and since the entire theory established upon oytological evidence involving only 15 open-pollinated seedlings of the variety, Bramsly's Seedling, it seeraed that more oytological evidence on vigor in exceptional v-:e&k seed­ lings would be very valuable. - 19 -

PURPOSE OF INVESTIGATION

The piirpose oT this investigation was to deterrftine v/hy

varieties^ suGli as Hibernal and "Virginia Orabjprodiioe v.-eai: and

undesirable seedlings and Vvby varieties such as Whitney and

/iflias 550 produoe strong and desirable seedlings. In pursuit

of this xmrTjose the investigsitor tindertoolc;

1, to determine the ohroffiosonie count and pollen

foriiiation in Eibernal, Virginia Grab, Whitney, iuiies

550, 8Jid any others ¥;hich might assist in the develop-

xaent of a more coisiplete unusrstanding of the behavior

of the first four varieties as seedling producers;

2, to determine ^Aiiether Hibernal, Virginia Grab,

Whitney and Arnes 550 veould set fruit in reciprocal

crosses •with diploid and triploid varieties;

3, to geriaiaate the seeds resultinj'? from these crosses

and ascertain the Ghromosome number of the resulting

seedlings in fin effort to determine if vigor is associ­

ated -'.vith chromosorae nuiiaber as postulated by Darlington

and Moffett (7). - so -

REVIEW OF LITEMTURE

Kobel (15) conducted tlie first cytological investiga­ tion on the apple. In a series of papers, he published counts on S8 horticultural varieties and 10 species (16, 17, and 18).

He found that the varieties v/ere divided into tvra groups; n.araely, (l) Varieties with normal reduotion alvisiori, and (2) varieties x^/ith abnoriasl reduction division. Thirteen of the investigated varieties belonged to the first group and fifteen belonged to the second group. The 2n chroriiosome count of the varieties "belonging to the first group v/as 54, v/hile in those varieties belonging to the second group it varied from 36 for some to as high as 51 in others. All the species with the exception of Malus Kalliana belonged to the first group and had an n chroraosocis number of 17, Malus Halliana had a 2n count of 47 -49 and v;as irregular in its reduction division.

Shortly after Kobel (16) published his first work, Shoe­ maker (45) found that normal pollen development in the variety,

Delicious, is associated with its ability to function satis­ factorily either as a pollenizer or as a parent in fruit breeding or stock investigations. In contrast, he showed that the variety Stajnnan Winesap, vvhich has abnormal pollen developnient 5 is lacking in ability to function satisfactorily as a parent in breeding or stock investigations. He stated that Delicious has 14 pairs of chromosomes and that Stayman v;inesap lias raore thfin 28 chromosoiaes in the Sn form.

About the seotie time, Rybin (41) published on rune species and one horticultural variety. He found soae of tiie species to be tetraploid, (lialus Sargentii 64-69, M. Toringo

64-71, and xMalus coronaria var. ioensis C. iC. Sclineid 65 chromosoraes). Seedlings of these species vere used in laafcing the above counts. The one horticultural variety was a diploid

(Tchulanovka).

Later, Kybin (42) Investig&ted the sosiatio divisions iu root tips of seedlings belonging to 5G varieties of the cul­ tivated apple, as v;ell as the reduction division in the anthers of nine varieties, lie found that the somatic number was 54- iiQ all varieties. liiight of the varieties had regular reduction division, normal pollen and & diploid number of S4 chroaosomes. The ninth variety had irregular reduction di­ visions, abnormal pollen and a trip-loid chroniosoine number of

51. This was the first definite recorded case of triploidy in iapples.

Maney and V/elter (30) foujGd that Malus ioeasis had 14 pairs of chroiuosomes and that a Mercer County seedling had a variable nurabcr ranging from 15 to 15 pairs. They noticed many cases of abnormal development in the Mercer Goiuaty seed­ ling. These abnormalities took the forxa of lagging chromosoraes on the enuatorial plate, v;hieh later on formed nuclei of their own. As a result, it was not unusual to fina five or six jnicrospores in the tetrad sta.<:^e.

Ghroaosonie counts for seven apple varieties were report­ ed by Heiltoorn (13) in 16S8. Six varieties ivere diploids

(2n = 24) and one, Gravenstein, was reported to be aneuplold

(Sn « 45). Kov/ever, it is probable that the laat variety is a triploid since Kobel (18) found it to have 45—16 and Kebel

(36) reporte^i. a somatic count of 51 for it aM seven of its bud sports.

lebel (24) investi^^ated tweuty-nine apples representing nine varieties anfl tv^enty species. He found the haploid COB]- plement in Malus was 17, and that diploid, triploid ano tetra- ploid forms 'ivere present. Kobel v;as soaewhat doubtful about the identity of the tvra species which he reported as triploid

(Malus speotabilis and M. prunifolia). He v;as inclined to regard the trees from vMch he took his laaterial a:; bein;? wrongly nacied. It appears th;jt his surmise was correct since

Hybin (41) has reported both these species as diploid and 3sx

(44) has reported M. prunifolia as diploid.

••In a later paper ffebel (56) reported 18 diploids and thirteen triploid s. A:aong the triploids were seven sports of

Gravenstein. Kebei (37) published a suiTtmary of, his previous papers in 1950 and induced with this a record of new chromo­ some counts v.hich he had made. These nev: counts included 4 triploids, 4 diploids and one ansuploifi (the last he question­ ed).

Thirty-one vsrieties and seventeen seedlings of cultl-

Yatoti apples ivere exanined by J)Rriiiigtoa and Lfotfett (7).

They raade both so;^atic and laeiotie counts end. coiifiraed Hybin

(42) and Kebel's (37) statemeata that the Iiaploid niaaoer in the app'le v/as 17, and thf.t sppie x^arietles fall into two larf-'^e groups, diploids v.ith 2n~ 54 and., triploids vrith Sn-- 51.

Evreinoff (10) in 1951 found that the haploid nuirAer

vvas not 1? for ell varieties examined,. Instead of 17 there

Wire VBrieties; with 12, others with IG, IS, 20, and 24 respect­

ively. Further he •jiaintained thi^.t Hybin was inoorrect vrfien

he gax'e 17 as the haploid number, and that Shoemaker's (46)

'/fork on the variety, Delicioias(Shoemaker gave this variety as

2n = 28) practically confirned his theory that there existed

in the apple various groups v/ith constant chronic somes niHribers

ano thv^t these differences could be used to erplain the vrri-

stion iu external oharaotei-s taetvreen apple varieties, and

finally how the varieties themselves ori.^iuated. Further, he

endeavored to show that his series of chro}aosoEie numbers in

the apple veere analogous to thoso in Chrysanthemum and Crepis.

Afliong the latest publioations dealing vlth chromoso?,ie

counts in apples are those by Sax (44 and 43). He made

chromoGOiae counts on 15 species. In all cases the species were either fiiploio., triploio or tetraploid. iineuploids vvere not observed.

Moffett (32) in an excellent paper on the Pomoideae adds chromosome counts on three horticultural apple varieties.

Also he examined the various groups of Poraoideae, all of v/hich were orthoploid, having 54, 51, or 68 chroraosojaes. The dip­ loid, species were quite similar in that at the division of the pollen mother cell the separation of the cliroraosonies was usually perfect although sometimes there v/as a slight lagging of one univalent. Two types of triploids occurred, auto- triploids and allo-triploids. The latter were formed froa a cross between a diploid and e tetraploid and had very irregular divisions with numerous univalents while the former usually formed only trivalents.

In an exhaustive treatise covering fruitfulness in the applSj psar, plum and cherry, Katividade (33) reported d-iromo- some counts for 15 diploic and 6 triploid apple varieties.

His data were in agreement v/ith those reported by Kebel (57),

Darlington and Moffett (7), Hoffett (3S), and Sax (44 and 45).

Further, Moffett's data (52)- from diploid x triploid and triploid X triploid seedling studies supported Darlington and

Moffett (7) and Crane and Lawrence's (4) assertion that cross­ es having one triploid parent produce aneuploid seedlings which are lacking in vigor. - E5 - la a sUiciy "begun in 1932 and publislied in 1955 Kebel

(Z'8) gave oViromosoae ooimts on 103 seedlings v/liich v;ere pro­ duced tfirou.gii controlleci crosses betw'een Sn x Zn, 2n x 5n,

3n >: Srij and 3n x Sn varieties of apples. All the seedlings produced froia crosses having at least one Sn parent v/ere aneuploias. Only 16 out of 89 arieuploid seedlings v/ere classa- fied e,s liaviug vigor, while all the seedlings froia the diploid

X diploic crosses v.-ere classified as vigorous.

ka iaterestiiig report on the cytological behavior of 15 diploic; and 2 triploid, apple vcrieties in France was given by

Miefizyreaocki (Til) in 1933, He noted that diploid varieties v;ere almost alvvays regular and triploid varieties irregular in their reduction division. His gerraination studies on diploid and triploid varieties were in agreement with Kobel

(16), Shoemalter (46), Heilborn (13 and 14) and Crane and Law­ rence (4). MiedzyreKecld. (31) also reported chromosome counts for three Diploid Malus species.

In her study of cultivated varieties of apples» Roscoe

(40) in 1934 reported fourteen diploids and four triploids, i\n.euploid.s were not observed.

Heilborn (14) la a very complete report in 1S35 reviewed reduction division, pollen lethality, and polyploidy in the apple. He presented a very cotaplex pollen lethal theory, based upon Darlinrton and fcloffett's "secondary pol3r])loldy'" (7)

GhromosoBie constitution in the apple as an explanation for the - go - pollen sterility T.4iioh is foiind in 'liyloia apple verietiss.

This treatise and tiie previous literature reYiev; clarify several points Y/iiicIi can be seea by exaraining tables and 3 of the thesis. These points ere na^Tiely:

(1) The cliromosoae constitution of the apple is, haploic: s 17 and diploid, triploid (and one tetraploid) varieties are found.

The aneuploids reported by Shoemrdcer {46), Kobel (15), Maney and VJelter (30) and Heilborn (15) vexQ not true aneuploids but •. fsr© in reality either diploids or triploids in Vvhieh the counts v;ere based only on reduction division figures. Since the ohroEaosorfles in,these fi/^ures are exceedingly sraall and often massed in /-roups it is very difficult to secure ortho- ploid counts. In fact, even in somatic plates this same con­

dition is encountered, but to a less extent. A second reason for these aneuploid counts is perfectly evident. Moat of these investigators did not have the best aicroscopic apparatus vdilch vould have assisted theai to make more definite, observa­ tions, (g) All of the investigators agree, that the diploid

varieties are practically alv/ays regul&r in their reduction

divirdon, (S) /ngain the litei'ature shows that the triploids

and the so-called "aneuploids" are irre.gular in their reduction

division, (4) Data from Darlington and Moffett (7), Crane'and

Lavrence (4), Bahl (6), Moffett (32) and Hebel (38) indicate that crosses having one triploid parent produce aneuploid seedlings. - 27 -

Table a. LIST OF GHIiQMOSOME KU?fflSRS IH HORTIGUl/IUEAL APPLE VAlilBTIES.

; •rt ;; * • M - » o 0 ' :f3p: © 'O , .065 ^ Ei ' tH o ••a: :tsS-: ISO +3 0 •H 0 .RO. d •p fl (D H a .Q H •^3 •© >D 0) •H 0

Mersleber Calville iU-::ero Alezander 34 Allington X~ippin 34 34 Annie Sllzabetli 34 Antonovka 34 iintonovlca gatmenitchlca 54 Apfel aus Lunov/ 34 Aport 54 Arkansav^ S4 Astraciian big trsmsp. 34 Astradian v/hite 34 54 Babiiskino 34 Bfdldwin (43-49: 51 51 Barlfevslcoje 34 Bauman's Reinette 56 34 Beauty of Bath 34 Belle de BOSICOOD 46 31 Belle Fille 34 Belleflower yellov:? S4 Belleflower x }:Citailca of Mitehurin 34 Belle Josephine 34 Belvl laliv 34 Beiuposta 51 Ben Davis Berner Eosenapfel 54 Blanche d'Espagne 34 Blenheisi Grange 51 51 Bohnapfel 49 Borovidnka-Borovv'i t sky v)4 Bramley's Seedling 51 - £8 -

Table 2 (contiixued)

© s3p; 'd O'iJ 'O U' •3 d -«-l CQ5 'd 654 CjO P o* o P(9 Ci !> H ,Q H •r4:^ ©a; 03 H H . su ^ Q)". •P ,^5 •H Wd, cj?; I!h M. 03 0 !>> © O cdcf moj o >• K W f£5 Pi(3' ci^ S

Branco 54 Bravo de Esaolfe 54 Burro 34 Calville blanc 34 Calville Lesans M 34 Calville ciu roi S4 Calville Grosshero?, o4 von Baden Calville blanche d'hiver ' 32 Calville rouge 32 Calville rouge d • autoinme 32 Calville rouge d'hiver ' 32 S4 Coiiioesa .fina S4 Canadian Eeinette 51 Canaille Sinap 34 Carlisle Pippin 34 Carneira 51 Gasa Nova, de Alcobaca 34 Cellini ^ 34 S4 Cerca 34 Charlomovi^sky(Buche ss of Oloenbiicg) 34 34 Coirnbra 34 Cox's Orange-Pippin 34 34 Cox's Orange-Keiriette 34 Cox's Poiriona 34 Crisison Beauty of Me'vV'- Brunswick S4 CriKison Braialej'' ^ 51 61 Damason-Reiaette (45-47) Danzigeer Kantapfel 34 Table g (oontiRued)

• • •H * t O o iiS+y Q) nii OQ? ''0 : "i-l N td d 4-IH H « H •rtisf ©a? 0 •H 0 •H © T-i H (U H ; a'ri •H 0 o fd 4^ ,o •H ,Q 03 (3) 0 0 a (U imct 0 P 0 •H {,,j a 14 P,C4 OA (tt-t M fU'H

Dash-Alma 24 Deacon Jones 34 Delicious (Shoemaker reported 2n = 28) S4 ])er Boelu'ier 24 Djir Hacizhi 34 Doigo S4 Douchin (falling Type II) Dudleys Favorite 34 Duchess Early Red Bird, Early Tiotoria S4 S fieri Eneroth's Klarapple 34 Esopus Spitsenberg 24 Espelho 51 FEila.vrater I'enouillet jaune 34 Fenoulllet roupe S4 Frosalcer 51 C-eante des Expositions . S4 Geheimerat Dr. Oldenburg 34 Gelber Hichard 34 S4 General Ton Haimjierstein ;54 Genete Moyle 51 Golden Reinette of Kursk 34 Golden Russet 34 Gold.enreinette von Blen­ heim 40 51 Grand Alexander {SS-34) Gragylling S4 Gronho S4 Gravenatein Banks Oris'ison 51 Gravenstein (45-46) 51 51 Grenadier 34 Table 2 (coiitinuGfi)

A 4J O CrP 0 CO -•d Cj,., N •pvf de. U it) EriiiU do. +3 o f-l do s4. 43 a a lo H a H* •r{3 (35 Qy 0 •H cr Ni ® •H H rtk 15-i ® ,£} cd;;. "t-i 'ji t!5 O dP! f-icd^ .0 > o" •H fr?' !^i3 Oi-T. iai £4 a?.

Grimes G-olden 54 34 G-ule-Peiilje Haliancl's Bi'unnsapple S4 Karapus Harbert's Seinette 45 Eurlbut 51 flusiiioder sapple 51 Irish PeacK S4 Jacques Lebel (49-51) Jaune de Metz (Mailing Type IX) 34 34 Jonathan 34 Kassaler-Reinette Kavlas 34 Jfentisli Godlin S4 Keswicic Cocllin S4 Kola 68 Lane' Prince Albert 51 34 24 o4 Lord Derby Lord Grosvenor , 54 Maeo'iiri 34 Maglemer 54 Manx Godlin 34 Mank' s lucheiiapfel 54 Margarstaapple red, 54 Mclntosb. Medina lifGlflp io 34 Melba 54 Melon 54 Menanaue: Japgerapfel (Hot Saiizler) 36 Mercer County Seedling (Haney and Vvelter reported £n =• 26-SO) Table 2 (continued)

: : : • * * •H . +3. o © "fri-p' CD 'd rooi'cf : N ca d PiO). (U <33 fcJ •USs'Ojo* -P 6 !H •H O ;Pt.O; Pi:+3 fl >> > H Id P H 00 0 -H 0 o;. •H H Q) :H ffi o 'd •H -Q .f-iti. cag:. 1^1 fH 05 (D (S f l> 0 ^•3 € 0 •aJC-fHCS- 0 0 •H

Milton 34 Minister von Hamraerstein 34 Muskat-Reinette 110 Kosiblot(Margille?) 51 Sev.-fane 34 Hev/ton V;'onfier 34 KonpareiliRoxbury Russet) 34 Nonsuch(Hailing Type 11) 34 Northern Spy 34 54 Odlins 34 Gland's Kungsapple 54 Old English Broadleaf Paradise(Mailing Type 1} S4 Ontario 34 Ontario Keinette 24 Oranie 34 Patte d'oie 34 I-^eurmain d' Adasi 54 Pfirsiohroter Somerapfel 24 J" xp •>> i54 P.

'C. 0®t5 ! p! Pi 6 fH id O 4J: •xi- o do ci •p. rt p r-f .H2 (» 0 0/^ f-f •Hi 'H r-t fi? H: d •p, •H p W liJ ^ I-. cd- f'r 0) & Cjff f^sS 0! w 3 fAcaOi~3

Kelnette Bleashiem 4-C Reinette du Canada (38-40} Heinette {-rise d'autoime 48 Heinette grise d'hiver 48 Reiaette grise cle ¥itry 51 Reinette pain de suore 24 Reinette Houge cl'Hiver 34 Reinette tr^s tardive 34 Reinette Zuccamaglio 34 Rev. Vs. Wilks 54 Reserla-Heinette 40 Rhode Island Greening 51 Ribstoa Pippin 4S 51 51 51 51 Rival M Roiae ^ S4 Rosenli^er 34 Eosmarin blano S4 Ross vile 51 Roter Siserapfel 47 Roter JiUiipfernapfel 24 Roter Stettiner 54 Sary-3inap 34 Sary-Tursh-Alma 34 Sclioner von Boskoop 46 51 Savstaholm 34 Signe Tillisch 34 Skvoznoy Maliv S4 Sommei'rarabour 34 Somergevsruraapfel 24 Sousa 34 Spatbluhender Taffetapfel 34 •Stlifner Rosenapfel (48-49) StUringe 34 Starlt 51 Table 2 (continued)

»» m 4 t • * « •H 0 • • * • HJ* O «) : : pip: : , 00. 'd . u S * --pik* rtffl* !w 0 X} U : ; SSO; 0 0 KO fj -P Q) r** > H ,0 tr~i d) T-1 0 W •H •H H , a) .iW , rtfi'. '!-( 0 O •H • ,0 • S-fCi* cdj5» m 0) & P 35 - (D • afl; O o M « m • g; «<£! Q.-:j ^ f=1 K

Stayman's V/inesap (Shoemaker reported 2n = more than 28) Stenlcyrka 34 Suislepper 34 Svanetrop 34 Tcherriogus 34 Tchulanovka 34 Tamara 34 Titovka 34 Toiapkins King 51 Transparente blanche 34 Transparente de Croncels 34 34 Transparente de Croncels X V;eisser Astrachan 34 Ti^i'enty ounce 34 Vaughan Seedless 54 Yitgylling S4 Vv'aflner 24 Wealthy 34 34 'iVarner' s King 4£ Washington 34 Weiclner's Goldrelnette 34 Wellington 24 Wellington BlooBiless 34 Winesap 34 Winter Banana ^ 34 Winter Golden Pearaiain 54,51 Winter Gray Reinette 34- Winter Magetin 34 Winter Zitronapfel (48-49) vVolf Hiver 34 'iioreester Pearmain 34 Yellow Hev^ton 34 Yollov; Transparent 54 54 York Ijnperiai 3^ Table 2 (continued)

« * e • t » 9 c •H . 4J. « * O • « 0) * rd : os: •W * iHM . •iw. ,0 f-! • 'oSH' do •p 0* h •H 0 ' flo. & >) !> H i=i A"- r—i* Odj a) •rl' CC INJ •H ffl •H H cs: H : v-( CD ^ •Xj +3 p ,C) TH ,0. wo. 1^. 0)

•Si ,Q H O ©. fjfl. 0 t>. •H Vrtf V.'J o fi-i W V-4 k'5« O'-f M. Pi 1?; • • • '

Saleiika Criraean 34 Zwansig Unacinapfel 34

1 Refers to ptoses 31 and 33. TriploicI as well as diploid seedlings \s/ere found. These varieties \Yere found to sho\v irregular dlTisiona. - 35 -

Table 2. LIST OF CHEOMOSOMI KUMBE HS I N ArI'Li

1 Species : Nebel: Sax :Rybin:Kobel:Darlington*

I « ; : :and Moffett

Mo adstrinf^ens 24 • sjiiurenxsi s 34 'A « angustifolia Michx. 66 24

» baccata ]3orkh. S4 24 24 ft • brevipes 24 rr a coronaria Mill. 68 68 ff 0 coronaria var. ioensia 0. K. Schneid 65 ft 6 Bav/soniana 24 If n eleyi(MiedEyrecki(31) rej orted the speciej3 Bn = 54) ?f « xloribunda Sieb. 54 34 34 fusca Schneid 34 ff 0 glaucescens Rehd. 68 68 ft » Halliana Koehne 24 47 ft 0 ioensis Britt, 54 ( Maney and 'I'elter (20)report- ed this species 2n=28) f! • Malus Britt. (P. Malus L .)M 34 and 51 11 • microijjalus 24 ?r • Niedwetskyana Dieck. 24 54 n • prunifolia Borlch. (? ) 51±1 34 24 f? • pruiii f01 ia mcroc arp a 34 iff • pumila var, paradisiaca 0, K. 3ohneid("ParadisG ") S4 34 it • puirlla var. praeoox G. £. SchaeidC'Doucin") 24 P. Hingo L, 34 M. rivularis 34

« robusta 34

« Sargenti Rehd. 34 (64-6"') rr • Scheidecksri 2ab. 24 S4 54 if a Sieboldi Rehd, 24 Jt » -Soulardi Britt, 54^ 24 • spectabills Borkh. 51'^ 24 (Mtedzyrecki(SI) Reported this species 2n« 24) M. sylvestris Mill, 24 24 24

Darlington and Moffett(7) report the apple under the generic name, Pyrus, the otiier authors (18){30) (SI) (34) and (41) under Malus. - 26 -

Table 3 (conulaueo)

:Neb el: Sax : Ryb in:'Kobel: Darl ington* : : : : :and Ivloffett

M. theifera Toringo Sleb (64-71) "» iumi ReM. S4 34

The species liave been tabiilEtted, as listed by the various authors except Darlington and. Moffett's (7) counts. KG attempt has been made to list the -various synonyms or to group the synoiijaas togethei*. 1 Bailey (1) lists the folloYdng species of apples: P. Malus, L. (Malus syl-vestris, Mill., M» doHiiauniSj D. C.).

?. Malus, var, paradisiaoa, L. (Malus puiaila. Mill,}.

r. Malus, var, apetala, Aschers. and Graebn.

r. Malus, var, Kiedwetskyana, Aschers, aiid Graebn. (P, Kiedwetskyana, Herasl.).

?. ioensis, Bailey(IsIalUB ioensis, Britt.).

P. Soulardi, Bailey(Malus Soulardi,Britt.)•

P. spectabilis, Ait.(Malus spectabilis,Bor}ch.).

P, pruiiifolia, ^illd.(Malus pruriifolia,Borkh.).

P. prunifclia, var. robusta,Bailey(Malus robusta,Rohd.),.

P. prunifclia, ver. Rinki, BsileyCMalus pruaifolia,Ter. Slakl, Rehd., M. Mngo,Carr., Pyrus lUngo, Wenz.).

P. ffiicii'oiflalus, Bailey(Malus raicrojaalus, Malcino., M. spectabilis, var. Kaido, Sieb., P. spectabilis, var, Kaido, Bean. ). P. anrustlfolia, Ait. (Malus angustifolia, Miohx.)» - 57 -

1 ?. ooronaria, Lo (Malus ooronaria, Mill.).

P. Hallirma, Yoss. (Malus Halliana, Koeime).

F. Halliana, var. Partaaanii, Bailey.

P. baccata, L. (Malus baooata, Borlch.}.

?. pulcherriraa, Aschers. and Graebn. (P. floribunda, Hort., not Lindl., Malus floribuncia, Sieb,).

P. pulchorrim, var. atrosanguiuea, Bean(r. atroseiiguiriea, Spaeth, 5 Malus atrosanguinea, Scliineid.}.

P. puloherrima, ver, Scheideckeri, Bailey(P. ScheiGeckerij Spaeth., Malus Scheideciceri, Zabel }.

r. pulcherrinia, var. j^iiiolCdana, Bailey (Malus iiriioldiaao., Sarg.}.

P. Zimiip Mats. (Malus Suai, Rehc.).

P. Sargenti, Bean(Malus Sargenti, Rehd.).

>?,. toringoidss, Osborn (Malus toringoidesj Hughes., P. transitoria, var. toringoifles, Bailey).

I'. Sieboldi, Eegel(Malus Siebolfli^ Rehd., I^ras and Malus Toringo, Sieb.).

P. fusca, Raf. (Malus fusca, Sclmeid., Pyrus rivularis, Dougl.).

P, Dawsoniana, Bailey(Malus Dav;soniana, Eehd.).

1^O First reported (34) 2n"S4. Later (35) as 51 but the last count was raade on juaterial which difl. not conform entirelji- to the described speoies. - 58 -

EJCPERIMEHTAL

Materials

The varieties Hibernal, King David, Starring, Araes 541,

Anies 550, Jonathan, Delicious, Grirties, Anieim, Staymsn, Whitney, and Virginia Grab were fcxenined cytolog;ically. Flov^er "buds v^-ere secured from trees in the experimental orchard et Ames, lotva.

With the exception of the three varieties, Ames S-il'''',

Vir?;inia Crab Gnu Ames 550, the above varieties ere listed and described by Fsaach (2), Aiues 541 is mentioned by Maney

(30) as being a large fruited, open-pollinatefi seedling of the Mercer County Crali. This is the only published descrip­ tion of A^nes 54:1, Yirginia Crab according to .Maney (25) is a definite variety discovered about 1885 at Muscatine, Iowa, among: some Hewes Yirginia Crab seedlingis. He stated,"It is propagated by root .^rafting like any standard variety and is not a {general line of rsiscellaneous seedlings as is so:-netimes conceived by those not familisr with the history of its origin."

/imee 550 is a seedling produced by the Poinolog-y Subsection,

Iowa Stcte Collerre, It ?.'as recognised as being an excellent

Tiie parentage of /unes 541 and hms 550 is: iuues 541 is an open-pollinated seedlinj^ of Mercer County Crab, &,nd Aises 550 is a seedling from the cross, Briar Sweet x L'ercer County Crab. 39 » stock pi'ocuoer Tjy Maney {27 and 28}.

Methods

During the aontiis, January, February and March, of 19SS, apple branv-^hes were taken froiu the college orchard and viere forced into "olooia in the greealic/ase. Later on, additional material was collected directly fros the orchard. The flower buds Vi'ere Itilled in Ofirnoy, Bouin, Allen's modificQtion of

Bouiu, Hemec, Kerpeehenko, and other variations of the general chroKo-acetic-formalin forraula. Of these fluids, Cej'xioy end

Bouin v^ere found to be most satisfactory for chromosome counts, although in some instances the other fluids gave very good figures. Hemec \'!&a very useful for general structure. Kar- pechenko and Bouin were found to be best for somatic fig'ures.

Gentian-violet-iodine, safranin-fast-green, safranin-gentian- violet and iron haematoxylin v;ere tested as stains. Since iron haemetoxylin was found to give the greatest differentiation betv;een the eytoplaaa and the chromosomes it was used through­ out the investigation.

Unsuccessful attempts were made to secure aoraatic counts on Hibernal, Virginia Crab, Whitney, Aiaes 550 and the various

Hibernal seedlings ahovm in figures 4 to 10, by forcing root cuttings in the greenhouse in the spring of 1233. The root tips from these cuttings v/ere so siaall and weak that the - 40 - individual somatic cells v/ere extreraely sBiall, These small, weaic root tips contained soiaatis figures but these could not

fee coimteci. accurately because the chroaosorries T/ere lying

closely together.

However, large and vigorous root tips were obtained from

open-pollinatea seedlings of the various varieties -hich con­

tained countable soaatic divisions. These v/ere secured laere-

ly by taking the seeds out of the fruit in Februai-y, 1533 and

eerainating them on moist blotting paper inside of a loosely

covered petri dish. As soon as the radicle energec, about

one-eighth inoh of the tip vms renoved for cytological study.

This mutilation does not kill the seedlings because they flourished v,!hen transferred to isoist sand.

Flov/er bud sections were cut from 6-10 microns in thick­

ness and root tips from 4-6 microns, Soiae 3000 microscopic

slides v/ere made during the investigation. These v'ere studied

carefully v

figures. V/hen these v.'ere found, the Haterial was examined

with great detail using a Spencer binocular microscope equipped

with a 90x oil-immersion objective and eyepieces having raagni-

fications of 15, 20, and 30x,

Pollen morphological studies v-ere raade on binucleate,

shed pollen grains in May, 1932. Similar pollen grains were

f!;ermlnated in petri dishes using a mediuia coraposed of 5% cane - 4-1 - sugar and Vh medium agar-agar and. 944 distilled water by. weight.

3)rav»ings were made v;ith a ca};aera lucida using the com­ bination of the 90x oil-immersion objective mth the 15>:, 2C;c or 30x oculars.

Cytolop;ical investigations

Whitney. This variety had regular reduction division.

Occasionally one lagging chromosome v/as seen in the first division anaphase (plate I, figs, 1 and 2, and plate Y, figs.

5 and 6). However, chromatin material was not observed out­ side the daughter nuclei following either the first or second division (plate I, figs. 3, 4, 5; 6, 7, and 15, and plate Y, figs. 7, 9 and 10). The bivalent chromosomes in most of the metaphase plates vvsre so close together that they could not be clearly distinguished and counted. Hov/ever, one fairly clear metaphase plate of the first division was counted vdth 17 chromosomes (plate I, fig. 8), Thus the diploid•chromosome number would be 34. In all instances, normal, four-celled tetrads v/ere found (plate I, fig. 9), Microspores \¥ere found

Vvith nuclei containing one, two or three nucleoli (plate I, figs. 10, 11, and 12, and plate Y, figs. 12 and 15). Uninucle­ ate and binucleate pollen grains were observed in the anthers

(plate V, figs. 14 and 15). Thus the first division of the pollen nucleus must take place inside the anther before the pollen Is shed. The generative nucleus v;as observed in the metaphase stage (plate I, figs. 18 and IS, and plate Y, figs.

17 and 18)» The division of tlie generative nucleus occurred after the pollen tube had grown considerably.

Pollen counts v^ere iiiade on dehisced pollen grains, as.6 per cent of these were eapty. One per cent ger.cains.tio-n v;as obtained with this variety.

Ames 550. This variety had regultir reduction division.

Figures 1, 3, 4, 5, 6, 13 and BO of plate II illustrate, this regularity. Seventeen bivalent chromosomes v.-ere counted at the first division metaphase (plate II, figs. S and 20).

Therefore, the diploid ohrosriosorrie number of the variety riuy,t be 134. Fifteen open-j^ollinated seedlings of this variety y/ere examined soHatically and each had a eomatic count of 34 confirraing the diploid count of 54 as obtained in the reduc­ tion division ,

ifigures 6, 7, 8, 9 and 15 of plate II illustrate the

Biethod of microspore formation in the apple. Instead of form ing a permanent wall iiaraediately after the first aeiotic di\-islon, the cell v.-all is not forined until after the second meiotic division, v;hen the four spores are deliraited siraultan eously by v/alls which are forraed by furrows developing imrard from the periphery. In the preparations, vacuoles did not seem to preces^dt'the furrov.'ing. This observation should be olieciced. using other stains and. killing fluids before being de-fiaitely accepted.

HoriUttl tetrads were observeii in all preparations (plate

II, fig. 13)» In a few cases, the jtiicrospores after being released from the pollen mother cell showed partial vacuola- tion. A high percentage of empty pollen .trains was observed, in all preparations (plate IIj fig. IS and 21).

Pollen counts were also made. Only 14.5 per cent e-mpty grains v/ere obser-veci. Fourteen per cent :?erraination v/as ob­ tained,.

Virginia Grab. This irariety vms very irregular in its reduction division (plate II, fig. 18, 19, SS, and SS, sM plate ¥, fig. 1, 2 and 3). The pollen mother cells Yi'ere normal in all respects. ChromosoBie counts ranging from 2S to

SS were made at fiiakinesis. Due to the siaallness of the apple ohroiaoaomes and the irregularity in size of the cluiaps these counts cannot be relied upon, iuiapliase counts '-ere jnade and. these also ranged fros 35 to 89 chr-omoHomes (plate III, fi£'.

8). Side viev/s of first division anaphase partially explain these counts. In the side vievvs the chrornosorries &re seen to divide at different times and ,in anaphsse the ni.ffiiber of lagging chromosoiiies is observed to be very high (plate II, fig. 18, snd plate U, fig. 1). In fact, there are usually one or two masses of chromatin lying toetv/een the two poles at late phase and vihich are not included rithin the tm daughter nuclei 'buti are seen in the cytoplasm during the telophase stage {plate Y, fig. 2).

In the second division anaphase the nusber of lagging oliroffloaoaes vvas nearly/ as numerous as in the first division anaphase (plate II, fig. £3, and plate V, fig. 5). rracticallj every division shov/ed from oris to five lagging chroraosora.es.

In the telophase of both the first and second division distinct masses of chror/iatin could be observed in the cytoplasra outside the da'oi'hter nuclei (plate II, figs. 19 and and plate Y,

.1•f 1 r,' O 1I c

Tetrad forraation appeared to be noraal in that 4-celled tetrads Vvere formed (plate II, fig, 25). In some instances, vacuolated Biiorospores were formed. This must have occurred betv/een the time of tetrad formation and the final rolease of the microspores froni the pollen another cell wall.

^'ollen counts v/ere made on dehisced pollen grains. Forty per cent of these Vvcre found to be empty. Only one-fifth of one per cent germination was obtained vdth this variety.

Hibernal. Lagging chromosoiries were observed in nearly all first division figures (plate III, figs. 1 and 2, end plate

V, fig. 4). Theso le^pging chroiQ,oso;U6S failed to be included in the reorganised daughter nuclei (plats III, fig. 3).

Figures of the second division anaphase v/ere not available but this division also must show lagging chromosoraes since second - 45 - division telophase figures show chromatin masses in the cyto­ plasm outside of the daughter nuclei (plate III, figs. 5 and

6). Counts v/ere Biade of first division metaphase figures but these were variable In number, since hi, tri, and other mul­ tivalent associations were comiaon. Figure 4 of plate III shov/s a first metaphase \\dth E4 chromosoffle bodies, three of v;hich have forraed a sezivalent. Again in fig. 7, plate III, a first division anaphase figure is shown vdth 24- chromosomes at one end and 24 or 23 chroraosoroes in the other. There are three small chromatin masses in this plate, one in the lov/er figure and two in the upper figure. These appear to be very small chror/iosomes. If this is true, the ciiromosome count of this variety must be 51 or 52.

The tetrads were very highly vacuolated in some prepa­ rations while in others they v/ere apparently nonnal (plate

III, figs. 9 and 14). In rare cases bodies which took the chromatin stain were distinguishable in the early uninucleate stage of the microspore (plate III, fig. 12). '

Approximately 37 per cent of the shed pollen Vi?as empty and only one-tenth of one per cent genuination was obtained.

Anisim. This variety v/as regular in its reduction division. Seventeen chroiaosomes v/ere counted at second raeta- phase in one figure and two sets of 17 univalent chromosomes v/ere counted in a first division anaphase figure (plate IV, figs. 1 and 2), Therefore, this variety is to be accepted as „ 46 - a diploid v;it.h Z4 ohi-OKOsomes. Starking. Both first and second division metaphase figures 6ho\ved 17 ohroBioso.rGeg (plate IV, figs. 3 and 6). So^ne-

times the second division metaphase figures v^ere difficult to

count, since there v/as a premature separation of the chromo-

sorae halTes for the following ansipnass di-visioa (plats IV, fig. S).

Ty.'o seedlings of Starring v;ere examined somaticslly and

in each case a diploid count of 34 was olDtained.

Delicious. The entire reduction division vi&s regular.

This variety shov>'ed mostly 16 Toivalent chromosomes in the first

division metaphase figures. With difficulty some of the raeta-

pha.se plates can be broken up into 17 pairs. The preceeding

variety, Starlclng, s bud sport of Delicious j is a diploid with

34 chroraosoEaes. Bud sports have been found in G-ravenstein to

have the same chromosome constitution as the parent variety

Nebel (37). It is generally recognised that Starking has

darker red color than Delicious, colors earlier in the fall^

hears earlier, and has a soaewhat stronger tree structure.

All of these characteristics point to a chromosonial change

analogous to those experienced in Droaophila v.-here on© mutation

often affects laore than one external chara.cteristic. Hence, it

seema that v.'hile only 16 bivalent chromosomes can be distin­

guished in the majority of the first metaphase figm-es, the - 47 ~ true count is 17 bivalent c]iromoso:iies. Still further evidence was secured from four open-pollinate(3. seedlings of Delicious which liad, a soiQutio count of 24 cbroaosomes. This conclusion v/as later confirraed by Roscoe (40) v,"ho has reported 1/elieious as a diploid with S4 chromQsomes.

Shoemaker (46) no doubt deterfflinec the count as 28 because he did not study somatic figures and took clumps of several chroraosomes in the metaphase figures to represent one cliroinosoirie pair,

iuna David. King David is a diploid v-iith 17 chroaosoiries in the haploid condition. Second division metaphase counts were niade showing. 17 bivalent chromosomes in each figure(plate

ITj fig, 7). The reduction division processes were regular.

Jonathan. All reduction division processes were ref^alar in this Vfjriety. A second division anephaae plate was ob­ served which shov/ed four sets of 17 chromosomes (plate 11, fig, 2;. Therefore, Jonathan is a diploid with 34 ohroiaosoBies.

Stayaan. This vsriety was irregular in its reduction division. Lagging chroraosomes v/ere observed in practically every first division anaphase figure. Eassos of chi'omatin were observed in the cytoplasm at the second division prophase.

In one clear first division anaphase figure, 30 chromosomes were observed in one nucleus and 27 in the other (plate IV, fig, 4). In a clear second division metaphase plate one - 48 - figure showed a count of 24; the other figure Vvas uncountable

(plate I?, fig. 5). Therefore, the sonatic count for St&yiaan auBt be at least 47. This is considerably higher than Shoe- malier's (46) report of nore than 26 chromosomes. Eleven open- pollinated seedlings of Stayman were examined soKatically,

An exact chrozaosoae count could not be raade on all these seed­ lings because the chromosomes lay so close together but ap- proxiraate counts were made. The first seedling had 44 or 48, the second, 40, the third 38 or 40, the fourth 36 or ?8, the fifth 42, the sixth 40, the seventh 38, the eighth 40, the ninth 40, the tenth 46 and the eleventh 39. The irregular condition as seen in pollen iaother cell figures and the aneu- ploid nature of the seedlings seem to Justify the conclusion that Stayaan is a triploid vlth 51 chromoaoaes,

.toes 541. This variety was irregular in its reduction division. This is in agreement with the observations of Maney and Welter (20), Another interesting fact is observed in the preparations. Maney and welter (30) reported the presence of iTiicronuclei in this variety. The preparations observed con­ firmed their findings. In the other varieties, wherever chro­ matin \vas seen in the cytoplasm, it seemed to be onI,y in individual chroiaosomes, but in this variety there seemed to be an accumulation of sever&l chromosomes in each clump of chro-

?natin (that is, the si?^e of the clumps was larger than one - 49 - ciiromosoi-ae). Accurate coiuits on this variety hs-ve not been made.

This V'riety raust be triploid rdnce stanciard varieties with an axieup.loid chromosonie constitution do not exist in the apple (Darlington and Moffett (7).

Grimes. Diakinesis figures vith 1? chroiiiosomes were ahunclant. Also, seoond dix'-ision laetaphase plates occurred with 17 bivalent chromosomes (plate lY, fig. 8}. Ifence, it is concluded that Griiiies has a fliploid, chr0rrj0S0;n,e nuiaber of

O'it

Chroriiosonial irregularities such as la^::ging were not seen in any of the preparations. Only normal tetraos v;ere observed.

Table 4 shows the relative amount of lagging in anaphase figures of the first division iu different apple varieties.

It illustrates hovi' the triploid varieties, Hibernal, Tirginia

Crab and Stapian, show lagging chroaiosoaes in practically all spindles, Tvhile the diploid varieties, Ames 550, Vihitney,

King David, Jonathan, Fyrus Baccata, Starkiag, Delicious and

Salome show scarcely any figures with lagging chromosomes. - 50 ~

Table 4. GOMP/iEISON Oli' GBSr:n?lD NORiilAL AMD yaiOm'M/' PIRi^T DIVISIOS AITAPIiASE FIGuI^ES IN DIPLOID mi TRD-LOir VMi'imiES,

Variety : HTiaiher of anaphase filiures : Total : ''Morraal" : ^Ahnormal"

Hibernal (Sn) 49 1 48

Stayman (Sn) 46 1 45

Virginia Crab (5n), 90 g 88 itfues 550 (2n) 15 12 3

Vihitney t2n) 50 43 V iiing David (Sn) 24 Oui0^'. 1

Jonathan (3n) 51 49 2

Pyrus baccata (En) 50 80

Starring (3n) 100 100

Delicious (En) 51 51

Grimes [Zn] f-uon V 20

Salorae (2n)-'- 50 50

1 Its cliromoBome behavior would, indicate that it was a diploid. - 51 -

Again in table 5 the diploid •varieties are shOYai to be almost laoicing in lagging chroiaosomes in the 2na division, vMle the triploid variety, Virginia Grab, shows lagging chroi-iosoKes in practically every spindle.

Table 5. G0:.3'AKI30N OF OBSKRViSD KOHIaAL Mi) "ABHOitMAL" FaCOilD IJITISIOK' AN./IPHASE FIGUEBS IK DIPLOID .\KD TRIPLOID VASIETISS.

tl'Iumber of gad division anaphase fii?ures 1'ototal : "Koraal""" ; ''Abnormal" i^JJl8S 550 (2n) 100 100 0 i/hitney (2n) 100 100 0

Jonathan (2n) 100 97 3

King David {2n) 23 21 S

Virginia Crab (3n) 100 4 96

Salome [Zn]'^ 8 8 0

.Salome'vS chromovsorrie behavior v/oulfi indicate thet it \vas a diploid.

From these tables it is evident that the triploid oon~ dition in the varieties. Hibernal, Virginia Crab, aiic. Stayaan is invariably associated with irregular division and. lagging ohroffiosoraes, while the diploid varieties, /iiaes 550, Whitney,

Jonathan, King David, Salome, Pyrus baccata, Grimes, Starking

{ind. Delicious are regular in their refiuction division. - 52 -

Table e« OOM:>AitISOH Or TRI KUIaBEH Ml) 'Pmomusm OF BiFTI AKIJ "KORm" OR FULL GEAIKS V^riHIH TliE AOTHKRS OF ms 550, Mlimi, ECBEENAL JUIP VIRGINIA GHAS.

~ ^ ' Pollenl^i^ns Variety : Total : Jitepty :"Korfflal"or full '.Mutaber obse-r^^edrPt-.T cent: I-'er cent

Ames 550 (Sn) 214 14.5 85.5

Whitney (Sn) 301 S8»6 71.4

Hibernal (Sn) 175 27.1 65,S

Virginia Crab (3n) 74 40.5 5S.5

Table 6 siiows that all four* varieties possess a large percentage of empty pollen grains but that the triploia

varieties have a iiuch higher percentage than the diploid

VBTleties. Fruit setting in reciprocal crosses involving the triploid varieties, Eibemal and Virginia Grab

Tables 7 and 8 shov/ the rejnjlts of crosses involving four varieties, Hibernal, Virginia Crab, Aaes 550, ana Whitney made during the years 1931 and 19S2.

Vt5ry feviT crosses v,ere successful in the spring of 19S1 because of a killing frost at pollination tiirie. Over 4200 flowers were emasculated and cross-pollinated froai vjhich .only four IVhitney fruits H-ere secured, v/hioh bore t?/enty-siA seeds.

These v^fere germinated in the spring of 1958, sixteen being viable. Fifteen of these seeclliiigs v^erc alive on October 15,

1952. Table 7 shows the data on these four fruits 8.nd their

Table 7. FRUIT SETTING II CROSSES BKTV/IEH WHITNEY?, /J?D AliES 550

^: r ; Tljlo. or"seeci- Breeding:Parentage^: Flovvers : Seeds :Seedlings:lings alive Number : ;pollinated;obtained:obtained :Oct. 15,195S

31701 Whitney x S60 16 10 9 Araes 550 (Sn X 2n} S170S V/hitney x 418 7 4 4 Duchess (2n X 2n) S1704 Whitney x 1S3 3 2 2 G-reen Svv'eet (2n X 2n?) All seedlings alive on October 15, 1^32 possessed, a high degree of vigor.

The 5parent is Vr'ritten first in each cross. - 54 ~

Table G presents the results in seed produotion and fruit setting of the cross-breeding carried on in the sirring of 193S. This table shows thet; (1) Yirginia Crab set only

4 fruits out of 465 cross-pollinated blossoras: (2) Hibernal set no fruits although 1,141 blossoms were croBS-pollinated;

(S) Mies 550 set 9 fruits froia 1,323 oross-pollln.ated blossoms; (4) Vvhitney set 387 fruits out of lj401 blossoss;

(5) reoiprooal crosses betAveen Hibernal and Virginia Crab v/ere complete failures; (6) where large enou^'h numbers are available it is seen that diploid x triploid crosses are de­ cidedly more iinsucoessful than diploid x diploid crosses. - 55 -

Table 8. FHUIT 3ETTIHG JM CR03SSS IKYOLVIHG SOiffi DIPLOID AID TEIiLOID YI\kim'IES ffl 1932, SHO'iVIHG KUIaBZR OF FLO^vlRS FOLLII^ATEI), .ASD PERCEi^TAGS OF I'LOVfEHS SBTTIKO FBDIT, TOTM. KUiffilR OF SEEDS SECURSD .AM) A7EHAGE MDIIBEK OF SESPS 3''FR IlxUIT.

Breed- rarentage*; Number Suraber:Per oent:Totel :Average ing s blossoi'fis fruit :pollin- ;number :number • number « pollin­ set ;ated ."seeds :seeds : ated :flowers 1 :per fruit

: :setting * a • :fruit «

32010 Whitney x 397 170 43.8 1,S53 7,4 Allies 550 (Sn 7; 2n) 52011 Whitney x 240 4 1.7 S S.2 Virginia Crab (2n X 3n) SSOIS Whitney x 487 3S 6.8 124 5.8 Hibernal (2n X 3n.) 52015 Whitney x 277 180 64.6 1,292 n/ • W9 Duchess {S,n X 2ii) 32015 juaes 350 x 425 6 1.4 S2 5.4 Whitney I3n X 2n} 32016 Ajiies 550 X 430 3 0.7 S3 7.3 Virginia Crab (En s 3n) S2017 Ames 550 x 470 0 0.0 Hibernal {En X 3n} 3S018 Virginia 192 4 2.1 12 2.0 Crab X Whitney (3n X 2n) 32019 Virginia 168 0 0.0 Grab x Hibernal I3n X Sn) 32020 Virginia 105 0 0.0 Crab X Araes 550 (Sn X 2n) - 56 ~

Table 8 (continued)

Breed­ ParentagerrKumber Humbsr ;Per cent :Total :Average ing ;blossoms fruit rpollin- :nuiaber:number number :pollin- set ;ated :seeds :seeds :at0d :flowers : :per fruit

; :setting * « : ;fruit * [

32021 Hibernal s 458 0 0.0 Whitney (Sn X 2n] 3208S Hibernal X 551 0 0.0 Mqb 550 (5n X 2n) 3S023 Plibernal X 352 0 O.G Tirginia Crab (3n X 5n) Totals 4,350 4E2 2,885

The female parent is v.Titten first in each, cross. In 195S cross-pollination of Maes 550 were laostly failures. This variety blooras much earlier in the spring than the other varieties used in the fruit breeding Vvork. In 19SS the weather conclitious during the .Inies 550 blossoming period viere unfavorcible for pollination. Temperatures v/ere lovv and scarcely s day i^asseo: vdthout soiue precipitation. These con­ ditions explain v.hy this variety aet so few fruits in 1932 v/heTi used in crosses involving diploid varieties.

As the weather conditions v^ere favorable, when the other tliree varieties v:ere pollinated, it seems fair to conclude that some factor other than v.-eather conditions was responsible for the poor results whenever Yirginia Grab or Hibernal was used in the crossing vrork.

Table 9 showa a comparison of the three varieties. Vir­ ginity Crab, Hibernal and /uaes 550, in respect to natural set in 1932. Prora this table it is self-evident that the diploid,

Ames 550, has a much higher natural set than either of the triploids, Yirginia Grab or Hibernal. It is also evident that

Ames 550 sets a higher percentage of its blossoipis than the standard diploid varieties where usually 3 to 5,4 \vill produce a normal crop. More than likely this characteristic is in­ herited from Pyrus baccata. It is true that there may be physiological as well as chroffiosoxaal factors involved in the fruit setting of these varieties. Hibernal is a large apple, while both Virginia Grab and kines 550 have sriiall ifruit. Eovi- ever, even if due allowance is made for thsse factors, the differences are so large that it seems that the ohromosoiiial factor may be the main one involved.

Table 9. COWPAKI30N OF THE APPLE VARIETIES VIRGIiaA CRAB, HIBEHK,ia. MD AliSS 550 IK RESri:CT TO K,ATURAL SET OF FHUIT IK 1932.

: Huxaber of : Hiuaber of :; Percentage Variety : blossoms : fruit set : of blossoms

Hibernal 560 8 S.2

Virginia Crab 300 17 5,7

.toes 550 165 100 60,6 _ o ..*•

ii.ll'ii.) V/0.W01jUoJ,

OliroTiiosoae constitution of cliploi6s

The preceeding data have shov/ii that the varieties, itoies

550, Vihitney, Jonathan, King David, Storking, lielieious, Anisia, and Grimes are diploids having 24 ohromosoraes. In -practically all instances the reduction division is regular altho\igh in a few divisions of Tihitney a slight lagging of one univalent was observed, after the other ehroaosoiaes had reeiohed the poles.

Chrosiosome constitution of triploids

The chroiaosome constitution of Hibernal, Virginia Crab,

Stayman and /laes 541 v;as not so definitely determined by counts of laeiotio figures. The large nuniber and, small size of the apple chroTiiosomes interferred with counting in spite of good preparations and high i!iag:nlfication. First division anaphase figures showed S5 to 29 ohromosoKies in Yirginia Grab, S4 and

S5 in Hibernal, and EO and 27 in Stayraan. There v;ere no counts of Ames 541.

In spite of failure to obtain full triplold counts of

51 ohrccTiosoiries in soiaatic cells or first division anaphase counts of 25 and S6, the suthor is convinced tlmt these var- itiss are true triploids and not aneuploids. These convictions are based on the I'ollowing facts;

1. All four varieties, had irregular reduction division. - 60 -

Z, Hibernal and Virginia Grab had a high percentage of empty pollen grains and the apparently norsiel pollen geriaiiiateci very poorly.

3. As parents in fruit setting studies Hibernal and Virginia

Grab proved to be unproductive aa shown in Teble 8.

4. Eleven open-pollinated ceefilings of Stayinan were aneuploids and the seeds obtained from Hibernal and Virst^nia Grab crosses failed to germinate. Also the Hibernal and Tirginia Crab seedlj.ngs observed by Maney (26, 27 and 28) ifere lacliing in vi;-or, (Figs. 1-3)» Finally, Hibernal as a parent produced only a fev,. weak trees . (Table 1 and. figs. 4-10). (Laiitz; 20).

5. "'hese varieties are vigorous grov.lng trees while all the aneuploid seedlings obsBrved as well as those reported by

Darlington and Moffett (7) Moffett (32) and Kebel (S8) are weaK growing.

6. Finally, v/here aneuploid varieties have been reported from meiotic material only, Kobel (18),Heilborn (IS), and Shoemaker

(46), and have been rs-exaained using both soiaatic and meiotic material these so reported aneuploids hav?^ been found to ba triploids. (Darlington and Moffett(7), Kebel (S&) and Hcscoe

(40)• Pollen studies of diploid and trlT:3loid varieties

The data shovved that the triploid varieties had a great­ er percentage of defeotivo pollen theji the diploids. Chroiao- somal irregularities in triploid varieties raay exijlain the - 61 - relatively greater ineffectiveness of their pollen, 'Ijut since diploid, varieties rilso have consiaaro'bi.e Aefective pollen, all defectiveneES cannot be attrilnited to chroiiiosomal ir­ regularity. v,'itii cir loic, varieties practically regular re­ duction division was observed vlth normal tetrad, formation.

Two other, explanations xmve been advanced as possible causes for riefeotive pollsn (1) eavironraental conditions curing pollination and (2) lethal fi;.ctor3. Heilborn (l;;) snd i'3cas:;yr2;ecld (:n.) observec that pollen sterility in the apple increesed v^'hen temperatures •••lere higher than normal. Heilborn concluded, that n.or»aal temperatures in v'^v.-eden v/ere too lovf to

ue a factor in diploid sterility. Hencs, Heilljorn (1? and 14) advanced the theory of pollen lethalo to account for diploid sterility. Heilborn's theory seeais logical because instances of pollen lethality have been reported by Sansoiue and Philp

(45). Heilborn uses Darlington and Moffett's (7) theory of

"secondary polyploidy" wliich postulates only oeven original

chroaosomes, P of xvhich are (ciiroaosoTaes A, B, and G) tripli­ cated and four of vrhich (ohroEOsomes D, 2, F, and 0) are

doublod to comprise the haploid chronio?or;ie set in the apple.

Hsilborn further supposes that there is one systes of pollen

lethals situated in the group of A-chroaogoviies and another

set of such lethals in the Ij-chroEiosoaes. Follen grains

having either of the pollen lethal systems in the hom.ozj'gous recessive condition shrivel and die. It is generally accepted that the present day Kjiple varieties rare hybrids developed froia inter-specific crosses.

Euhce, it might follow that they are characterised by ohrorao- soiiie constitutions possessing certein varying types of pollen lethal systems, and vdien varieties are crossed theso various lethal systeBis result in different levels of sterility.

The pollen formation studies resolved themselves into an inquiry into the causes of sterility, for which the author offers the follovdng explanations:

1. firjit, unfavorable environmental conGitions can and GO reduce fertility, possibly in a similar degree for all var­ ieties, es:cept those v/hose inherent chroffiosome constitution makes theia especially susceptible-j.

Z, Second, tiiere appears to be a set or sets of pollen lethal systems vhich produce pollen sterility in the apple when cer­ tain gene coabinationa are encountered in the haploid, pollen grain v/hether in diploid or triploid. varieties.

S. iVnd, finally, in triploid varieties, the unbelanced fmeu- ploid chroiiosone nu;iiber of the haploid pollen grain produces an additional and dominating crpl&iiation for the high degree of sterility in such veirieties.

Fruit setting in reol-procal crosses involving; the triploid varieties» Hibernal and Virginia Grab

The cross-breeding data shown in Table 8, agrees v.'ith that observed by Lentz (20) in that the triploid, Eibernal, - 63 - is an extremely poor breeding parent. It seems vdthout a doubt that the conflicting evidence £cv.3nced by Beaumont (S) and the experimente1 farm at Lennoxville, i^iiebec l2S; as to

Hibernal's behavior vms clue to their having a strain of

Russian apple v;liich had been named Hibernal but vvhich was cytologically distinct froa that strain used in this investi­ gation. The lack of germination of the Hibernal and Virginia

Crab seeds confirms the findings of Lantz (20) and Maney (26 aUU iCi / / «

In future cross-breeding progrejus involving triploids, little time should be spent on 2n x 3n crosses since triplcid pollen is largely ineffective. Generally speaking triploids should not be included in a practical breeding progrtmi since they are very likely to yield a deerth of results.

G-sneral

The behsvior of the Hibernal and Yirginia Grab seedlings, which are lacking in vigor, is explained by the thesis obser­ vations which shov; that,

1. Hibernal, Virginia Crab, Araes 541, and otajniian have ir­ regular reduction c.i•vision.,

2, The pollen of Hibernal, and Virginia Crab is lovv in fertility and germination,

S. Hibernal and Virginia Crab are unproductive when used in fruit setting studies (Table 8), » 64 -

4. Hibornal snd. Virginia. Grab seeds germinate very poorly

(ivtoney S5, S6, and 27), and (Laiitz 20),

5» The eleven open-T)ollinated seedlings of Stajnjian Vi'ere aneu- ploids.

These triploid varieties through their irregular re­ duction division produoe irdcrospore-s so lacking in vigor due to their unbalanced chromosoKie constitution that few of them ever reach the pollen f-rain stage in a viable conditlono

Very few that progress to this stage are likely to form sperms

and fertilize egg cells. Thus the effects of the unbalanced

chromoaome number are encountered in all stages of ropro- duction and growth of the seedlings- vdth a consequent mort&l- ity in the triploid seedling population at all stages.

]jiploid varieties, such as Vvhitney and Aiaes 550, on the other hand, in spite of the fact that they show some pollen sterility and soraetiiaes faulty seed development, regularly produce far more vigorous seedlings than triploids. This ability to produce vigorous seedlings is associated In the diploid verieties vrith such behavior as the data have shown, namely, (1) regular reduction division, (2) relatively low percentage of pollen sterility, (3) higher percentage of pollen

Germination the^an triploid varieties, (4) high degree of fer­ tility as mile or female parents In fruit setting studies,

(5) good seed germination, and, (6) orthoploid chromosome con- - 65 - stltution of open-pollins.ted seedlings from diploid var­ ieties.

While seedlings of crosses betv/een diploids are laore vigorous than seedliiif';.'? of triploids, %mriutions in vigor araong diploids tliemselves jmst be reccgniiaec.. Svifience has been shown l;y Lanta and liis Go~v.orkers (9, 19, 21, and 22}, and Zdgecoabe 18} that seedling profvenies iron diploids differ in their raean level of vigor.

It is suggested that cytologicsl studies dealing vnth the evolution of th© apple raight well take the forui of studies of seedlings from reciprocal crosses betvreen 3a x Sn, and 2n x to forms, using horticultural vairieties and Malus species in an attempt to ascertain v/hether triploifl forms could be produced. The cultivated vsriety, Kola, vhich is

4n should be used with 2n and Sn parents in an effort to produce ;3n types of horticultural value.

Hov/ever, since relatively barren results are secured from Sn cross-breeding programs, it would be v;ell to supple­ ment the above %'ith cytological studies of open-pollinated seedlings from 3n Varieties. To date no one hv=i,s reported the pollen mother cell development of an aneuploid seedling from a Sn parent due to the usual cause -- death of the aneuploic before fruiting age. This chculd be investigated for in- foriBation on reduction division in aneuploid seedlings. - 66 ~

The possibi?^ity of •utilizing triploics ir. a different

\vay from any thus far reported which idglit yield surprising results, naaely, the seeurinr of somatic doubli^ig of the chromosome niuaber in the 2n, En, axid in forms is also suggea ed, GrmB and Lav/renoe (5) point out that higher merafeers of a polyploid series usually present a greater range of selec­ tion Qong horticultural lines. Hence, if such doubling could, be secured the now useless Sn breeding varieties night well become excellent 6n varieties of extrenely high value in themselves. The asexual propagation methods used with the apple v;ouia raaice perpetuation of such varieties a very simple ana practical matter. - 67 -

SiUivMMlY

1, A cytological study was mde of twelve apple varieties

and their seedlings in &ii effort to explain why, HilDernal and

Virginia Crah produce seedlingts lacking in vigor, v/hile kn.es

550 and Whitney produce vigorous seedlings.

£, The diploid verietics, Vfhitney, Araes 550, Anisira, Starking,

Delicious, Sing David, Jonethan, and C-riaes, were found to he

Siis S-ij to have regular reduction division, high percentage

of norraal pollen, a relatively high germination of pollen, and

v;hen used in crossing with diploids to be productive,

5, The triploid varieties, Hibernal, Virginia Crah, Staj-inan

and .fcaes 541, Vvere found to be 2n as follows. Hibernal (at

least 49), Virginia Crab (at least 46), and StajTiian (at least

4?). No counts were made on Anies 541. Since these co\mts

were made from meiotic divisions only and since vigorous grow­

ing aneuploida are unlmovm these varieties are assumed to be

triploids vdth 51 chromosomes. They were found to hyve ir­

regular reduction civision, poor pollen geraination, a rel­

atively lov; percentage of normal pollen, and when used in

crossing vath either diploids or trlploids the crosses were

usually complete fuilures.

4. All open-pollinated seedlings irorii diploid Vfarieties v.hen

examined cytologioally gave a cbromosoae count of 2n=34 and vrere vigorous in growth. - 68 -

5o Ail cytologicol exsiainations on open-poilinated seedlings

I'i'ora triplC'id varieties shov/ed these seedlings to be aric-iiploid with a soHiatic ehromosoiae constitution bet\veen 34 eind 51.

6. Hence, tlie lack of vigor of Hibernal and Virginia Crab seedlings is explained on the basis of their irregular chromo- aome behavior during iieiosis, gametes being formed with an unbalanced Qbroraosoiae constitution, vrhich on union with the feraale paraetes produce aiieuploid individUEils, these seedling individuals being constitutionally laolring in vigor due to their abnormal chroxaosome constitution.

7. The varieties, Whitney and imes 550, produce more vigor­ ous seedlings than the triploids, Hibernal End Virginia Grab, because tlieir reduction division processes are noriaal end cons6(iuently the gamcites and finally, the seedling offspring when produced from crosses *i-dth other diploids are orthoploicl.

8. Finally, the data substantiate the viork of Darlington and

Moffett (7), Crane and Lavi'renoe (4), Moffett (32), Hebel (36),

Heilborn (14], and Dahl (6) in that, ehroHOSome nusber in apple seedlings is associated vdth vigor in triploid crossed where aneuploidy is a factor. ^SKPLANATIOK OF PL/;T:c {QajABTa liicida drawings)

i/iguxes 1 to 19 inclusive of 'iVhitney. 2CG0x except figin'e 19 whicli is 1300x.

Fig. 1. Sicle view, first late anapaase.

Fig. 2, Sioe view, first late anapha^je vvith one lagging

clironosorne.

Figs. 3-4:. First telophase figures; in fig. S the nucleoli

are alraost eqiial in si'/,e, in fig, 4 one member of

each pair is ueciaedly larger than the other.

Fi§. 5. Regular second anaphase.

Fig, 6. Early second telophase; siaall nucleoli and many ;nasses

of fused chromatin.

Fig. 7. Variable nuraher of nucleoli at late telophase.

Pig. 8. Polar view, first nietaphase plate showing 17

bivalent chromosomes.

Fig. 9. Normal tetrad.

Figs. 10, 11 ana IS. Hormal microspores with one, t\vo a-ic

three nucleoli respectively. {'Tithin the anther).

Fig. 13. Binucleate pollen grain; vacuolation in the cytoplasm.

Fig. 14. Side vie^v, second raetaphase plate.

Pig. 15. Side view, early second anaphase.

Fig. 16. Binucleate pollen grain.

Fig. 17. Empty pollen grain.

Fi;;^, 18. Section of pollen tube containing sice view of

metaphase plate of generative nucleus. Pig. IS. "a" shows location in the pollen tube of netaphase plate of fig. 18, about 15ux. - 70 -

PLATE I

•r:.>.:i T,'i\\y:::y;y rz "*••••'•«' •' /.".in » .fc' 4^ j,*

'it'i'-i'fcr•7 r J idMi Rj' '*>'••-'-'rV. !•

•Il{> l."C^/.H

12

'"•V '.•*•"*

IMirr. •••••wV t-i

16 17 Siif 18 19 2XPLAKATI0H OJ PLATS II {Camera lucifia drawings, 3000:c).

Figures 1 to 17 inclusive and 20 and SI are of Ames

550. Blgures 18 and. 19, and 22 to 25 inclusive are of Vir­ ginia Crab.

Fig. 1, 3it3e view first anaphase.

Fig. S. First raetaphase plate with 17 bivalents and one

fragment,

Fig, 3. First telophase.

Fig. 4. Side view second laetaphase.

Fig. 5. Side viev/ early second anaphase.

Fig. 6. Late telophase second division.

Figs, 7, 8, 9, 13. Formation of tetrads; fig, 7, pinching

in process of outside nails; fig. 8, three members

of tetrad Just separated; fig. 9, tetrad aho^Alng

two Kiicrospores slightly separated; fig. 13, tetrads

completely separated.

I'ig. 10. Kormal microspore; two nucleoli present.

Fig, 11. Koraal microspore; one nucleolus present.

Fig. IS. Empty microspore.

Pig. 14. Microspore released from pollen mother cell wall;

vacuolation evident; cell v/all has thickened.

Fig. 15, Binuoleate pollen grain; note thickened ccll v.'all.

Pig. 16. Uninucleate jjollen grain,

?ig. 17. Dehisced binuoleate pollen grain; cell vvall much . *1 thickened.

rig. 16. aiae viev/ first anapnase-;' four lagging masses of oliromatin. Fig. 19. Second prophase; four micro-nuclei in the cvtonlasm .

in process of outside y.alls; fig. 8, three raembers

of tetrad just separated; fig. 9, tetracl .shov.'ing

tvv-o raicrospores slightly separated; fig. 13, tetrads

oospletely separated.

Fig, 10. Kormal isiorospore; tv^o nucleoli present.

?ig. 11. Korifial microspore; one nucleolus present.

Fig. 13. S/Tipty microspore.

Fig. 14. Microspore releasocl from pollen motlaer cell wall;

vaeuolation evident; cell \'«ill has thickened.

Fig. 15, Binucleate pollen grain; note thickened cell vrall.

Fig. 16. Uninucleate pollen grain.

Fig, 17. Dehisced binucleate jjOllen grain; cell wall raich

thickened.

i/ig. 16. aiae view first anapnase'; four lagging masses of chroinatin. Fig. 19, Second prophase; four micro-nuclei in the cytoplasm . ?ig., 20. First anaphase plate vdth two sets of 1? univalent

chromosomes.

Fig, 21. Empty dehisced pollen grain.

Fig, 22. Second telophase; four chromatin masses in the

cytoplasm; vaeuolation present.

Fig, 23. Side view second anaphase; lagging chromatin masses

in hoth spindles.

Fig. 24. Second telophase; four nuclei present. Note lack

of stained m.embranes.

Fig. 25. Normal tetrad formation.

PLATE II EU'LAl'IATION OF PLATE III {Camera lucida drav/ings)

Figure 8, Virginia Crab, 2000x; the remainder of the plate of Hibernal; figures 13, 15, 16, lS00x,,the other figures SOOOx.

I'ig. 1. First anaphase v/ith five masses of lagging chromatin;

one Tiiass of chromatin at upper left hand of cell.

Fig. ,2. Similar first anaphase vdth two ls.gging trivalents

(?) and one lagging bivalent. llZ' 3. JSarly first telophase shov/ing five raasses of chro­

matin in the cytoplasa. 'The nucleoli are distinct

and take a very heavy nuclear stain.

Fig. 4. Polar viev/ first metaphase showing SI bivalents, and

1 sesivalent.

Fig. 5. Late second telophase shov/inj^ 3 masses of chroraatin

in the cytoplasm; one or two nucleoli in the nucleus.

The dotted lines indicate the "pinching in" process

v/hich precedes tetrad forrnation..

Fig. 6. An extreme case of abnormality in second telophase.

3 nuclei are formed; the fourth has failed to form

a nuclear membrane and the chromatin is released

into the surrounding cytoplasm.

I'ig. 7. Side view first anaphase showing 24 chromosomes in

one plate and S5 in the other. Note "a" this chromo­

some may be uni- or bi-valent. There are three very

small staining bodies in the cell which might be

chromosomes. If so this cell would show the triploid

count of 51 ohi'ciiiosomes.

Fifi, 8. VirEinia Grab: DOlar view first anaphase with 29

Fig. 5. Late second te3.ophase showing 3 masses of chrcraatin

in the cytoplasm: one or two nucleoli in the nucleus.

The dotted lines indicate the "pinching in" process

v/hich precedes tetrad formation.

Fig. 6. An extreme case of abnormality^ in second telophase.

3 nuclei are- formed; the fourth has failed to form

a nuclear membrane and the chromatin is released

into the surrounding cytoplasm.

Fig. 7o Side viev/ first anaphase showing 34 chromosomes in

one plate and 25 in the other. Note "a" this chromo­

some may be uni- or bi-volent. There are three very

small staining bodies in the cell which might be

Ghroiaosoaies. If so this cell would show the triploid

count of 51 ohroraosomes.

Fig. 8. Virginia Crab; polar viev/ first anaphase with S9 chromosomes..

Fig. 9. Tetrads with dense cytoplasm.

Fig. 10. Vacuolated microspore released from pollen mother

cell wall.

Fig, 11. Ghroraosome grouping in first metaphase an.d anaphase

plates.

Fig, 12. Microspore-ivith dense cytoplasm and distinct masses

of chromatin in the cytoplasfa.

Fig. 13. Binucleate pollen grain.

Fig. 14, facuclated tetrads.

Fig. 15. Empty pollen grain.

Fig. 16. Typical binucleate pollen grain of the apple.

PLATE III EXPLMAIiOK OF PLiVTS 17 (Camera lucida drav.lugs) Magnification 4000x

'Fig, 1. PsdB'ha. Second metaphase showing one plate •i'^ith

17 bivalenta. The other plate is massed and un-

eoiaiitable.

Fig. £. ilnisin. Slrst anaphase showing two sets of 17

univalent chroEiosones. One ,yet is in black, the

other in outline.

Fig. S. Starkin/?. Second aetaphase showing tx^vo plates, each

vdtli 17 chromosoaes. Hote the splitting of the

bivalents for the anaphase separation.

J'i£, 4. Stayiaan. First anaphase showing 20 ehro:aoso'.:;es in

one group and E7 in the other group.

Fig. 5. Staynan. Second raetai^hase showing 24 Ghromoso.i;ies in

one plate. The other plate is uncoimtable.

Fig» 5. Starking. First jaetaphase shoeing 17 bivalent

chroraosoriies.

Fig. 7, King Da,vict. Second, aietaphase showing two sets of

17 bivalent chroraosoiries.

Fig. 8, Grimes. 1 figure from second metaphfise shov/ing 17

bivalents.

Fig. 9. Jonathan. Second anaphase shovdng four sets of 17

univalents. - 76 - PLATE IV

C-Vi-AA,

.ys >•»-•>.•«•*»»'»«•

^"•:*-:S<&s'^;vi!-:^-VK'>!BHI

$isMS ^mdam^iim

•r.v,^i^5/.'.**.»^*.vi - ^-''VvAf--•

-i.v»/;',;i:>*

wP.yy. i •;•••; •'i''-'S siiills 0M0 ••iixfrf' EXPLAIWfflON OE J'LASE Y (PhotoHiicrographs 1450x except figures 16 and 17 v/hicli are 150j] fig. 1. Virginia Crab pollen mothGr cell shov/ing lagging diromoaorftes at first anaphase. Fig. a. Virginia Crab pollen mother cell; first late telophase. Characteristic chr-oHiatln iri8.sses in

the cytoplasm outside the nev/ly foriiied nuclei.

Fig» 3. Virginia Crab pollen raother cell; second anophase

with lagging chr0210s0s.es.

Fig. 4. Hibernal pollen raothex" cell; first anaphase vdth

nuir^erous la^^ging chromososes.

Fig. 5. V'/hitney pollen iiiother cell shoYvinc first anaphase.

Fig, 6, \Vhitney pollen jiiother cell; first anaphase with one .

slightly lagging chromosome.

Fig. 7. Whitney pollen siother cell; first telophase.

Fig. 8. ?(hitney pollen mother cell; norraal second anaphase.

Figo 9, Yvhitney pollen mother cell; norraal second early

telophase.

Fig. ID. Same as fig. 9 except that t\ra nucleoli have develop­

ed in some nuclei at late second telophase.

•Fig. 11. Three microspores released- from noriaal Whitney tetrad. Fig, 12, Whitney microspore vdth one nucleolus.

Fig. 13. Whitney microspore with two nucleoli.

Fig. 14. Whitney uninucleate pollen grain inside anther. Fig. 15. ffhitney binucleate pollen grain within the anther.

Fig. 16. Four Whitney pollen grains. i'is* J.7, "ii" ijigxiiries ths iccation' of the

sietgigwsaie j-iete tk® gsK&retlve nusl-eus •yjitiiiB

Fig. 8, V'/hitney pollen motiier .oell; normal second anaphase,

Fig. 9. Vviiitney pollen mother cell; normal second eej'ly

telophase.

Fig. 10. Sarae as fig, 3 except that two nucleoli have deve3.op-

ed in sorae nuclei at late second telophase.

Fig. 11. Three microspores released froa norraal Whitney tetrad. Fii", 12. Whitney aicrospore 7;lth one nucleolus.

Fig. 13. Whitney microspore with tvio nucleoli.

Fig, 14. Whitney uninucleate iJollen grain inside anther.

Hg. 15. Vihitney .binuoleate pollen grain Vvlthin the anther.

Fig. 16. Four \Vhitney pollen grains. i'ig. 17, "a'"- aiguUles the locution'oX the

issta;ph&s{& i l&te Gf the mn&r&tivt:! aual-evis the pollsa tubo. Via^ IB. lalarf^esent of the ares "a" shoeing & aide setephase

view of the generative nucleus. Note the spindle

arrangement.

Fig, 19. Normal mature pollen grain released from anther;

note tvi'o nuclei and inclusions.

Fig. 20, Starring pollen inother cell; side view firnt

anaphase,

- 78 -

PLATE V

IB LITSRA.TUR1 CITED

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Beaumont, J. H. Preliminary report on relative vigor of apple seedlings, Proc. Mer. Soc. liort. 3ci., 25:349-257, 1928.

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Genetics of garden plants, p. 56 and 65. Maomillan Co., London. 1934.

Dahl, C. G. Root stocks from seeds of known parents. Int, Hort. Congr'o 1950. p. 141-149. 1931.

Darlington, C. D. and Moffett, A. A. Priinary and sec-, ondary chromosome balance in Pyrus. Jour. Genetics. 22:129-151. 1930.

Edgecombe, 3. Vs'. Apple breeding; the effect of parent­ age on the relative vigor, grade, and corre3.a- tion relationships of crossbred apple progenies. Unpublished thesis. Library, Iowa State College, Ames, Iowa. 1931.

and Lantz, H. L. Apple breeding: Sta­ tistical analysis of apple breeding material. Free, ilmer. Soc. Ilort. Sci., 28:93-96. 1932.

Evreinoff, V;. A. i^tucle carjrologique comparee d6 Pommiers coiame base d'un System© pomologique. Rev. Gen. Bot., 45:474-491. 1931,.

Gibb, C. Report on Russian fruits. Montreal Hort. Soc. Proc., Rpt,, 8:17-89. 1883. - 80 -

» With notes on Russian apples Imported by the U. S. Department of Agriculture in 1870. Reprinted from Ont. ifruit Growers' Assoc., Rpt. -for 1885. Blackett Robinson C. Toronto, . Canada. 1884.

Heilborn, 0. Zytologische Studien iiber Eollensterilitat von Apfelsorten. Svensk Bot, Tidskr., 2S:185- 199, 1928.

Reduction division, pollen lethality and polj'ploidy in apples. Acta Horti Bergiani, 11:129-184. 1955.

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and Edgecombe, S, Vs. Apple Breeding: Some significant differences in the vigor and grade of crossbred apple seedlings. Proc, iiiaer. Soc. Hort. Sci., 27:289-295. 1931. - 81 -

and. lierrill, S. The vigor of Aatonovka seedlings. Free. Amer. See. Hort. Sci., 24:115-120, 19S7.

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Lyon, T. T. Report on the adaptation of Eussian and other fruits to the extreme northern portions of the United States. U. S, Dept. of Agr., Div. Pomology Bui. 2. 1888.

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.. Grov/th characteristics of a number of selected anple stocks. Proc. Amer. Soc. Hort. Sci., 27:94-101. 1931.

, Plagge, H. H. and Pickett, B, S. Stock and cion effects in top-worked apple trees. Proc. Amer. Boc. Hort. Sci., 33:332-335. 1936.

______and V/elter, "h A. Chroiaosome character­ istics of Malus loensis and one of. its large fruited forms. Proc. Araer. Soc. Hort. Sci., 25:115-118. 1929.

Miedzyrezecki, Oh. Etudes cytologique et sterilite du pollen chez le Pommier et le Poirier. Gompt. rend. Soc. de Biol., 114:1267-1271. 1933.

Moffett, A. A. The chromosome constitution of the Pomoideae. Proc. Roy. Soo. Lond., B, 108:423-446. 1931.

Natividade, Ch. A. A improdutividade en poraologia.

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Roscoe, Muriel V. The chromosomal constitution of certain cultivated apple varieties. Jour. Qenetics, 28:157-167. 1954.

Rybin, V. A. Cytologioal investigations of the genus Malus (preliminary account). (Snglish summary.) Trudy Frikl. Bot. ISelek., 16, 3:199-200. 1926.

. On the number of chroiaosomes, observed in the somatic and reduction division of the cultivated apple in connection iilth pollen sterility of some of its varieties. (English suiffiuary.) Trudy Prikl. Bot. i Selek., 17,5:120. 1927.

Sansome, W. and Philp, J, Recent advances in plant genetics, p. 44 and 51. F. Blakiston's, Son and Co. Inc., Philadelphia. 1952.

Sax, K. The origin and relationship of the Pomoideae. Jour, ilrnold Arb., 12:3-22. 1931,

. Gliromosome relationships in the Pomoideae. Jour. Arnold /irb., 13:363-367. 1932.

Shoemaker, J. S. Pollen development in the apple, with special reference to chromosome behavior. Bot. Gaz., 81:148-172. 1926. - 65 -

ACa;OWLE:I}Gi^HTS

Tile v.Titer v/ishes to exgvess his deep appreciation to those members of the Jjepartments of Horticulture and Forestry, of Botany, and of Genetics vflio aided in laaking this investi­ gation.

Special credit is due to Professor B. S. Pickett, Eead of the Depe.rtraent of Horticulture and forestry, v/lio raade it possible to study the jjroblem and vAo generously cooperated throughout the investigation, and to Dr, .Jolm K. Martin for his splendid cooperExtion end helpful suggestions on the cyto- logical phases of the investigation- Farticular thanks are due to Professors T. J. Ilaney and H. L. Lantz for their oon-

Etructive advice and cooperation. The writer also vdshes to aclaioY/ledge the helpful assistance of Br. 1. V/. Lindstrom,

Head of the Departraent of Genetics, in the genetic phases of the investigation.

finally, the writer is deeply indebted to Professor r. Vf. Brodriok, Hejid of the ijepartment of Horticultui'e and

Forestry, Professor Y. W. Jackson, Head of the Ueparti'.ient of

Botany, and Professor A. T. Slders, Assistant Professor of

Agronomy, all of Manitoba Agricultural College, Winnipeg,

Manitoba, Canada, Ydio emphasised the need for more cj-tological investigations in fruit breeding prograxas, and w'ho encouraged the \a-iter as an undergraduate to follovir this line of graduate study.